Method of modifying glucose activity using polypeptides selectively expressed in fat tissue

ABSTRACT

Isolated omentin polypeptides that selectively express in omental fat tissue and methods for the use of the polypeptides. The polypeptides can be used in a method for modifying insulin action and/or glucose metabolism in an animal. The polypeptides can be used to promote glucose uptake by animal adipocytes and other cells, tissues, and/or organs. The polypeptides can also used to provide a therapeutic treatment for diseases of or related to glucose metabolism and adipose tissues. The polypeptides are also incorporated into diagnostic tests and testing kits for diagnosing or detecting a disease or condition involving animal tissue that contains, uses, or expresses the polypeptide in an animal suspected of having the disease or condition.

CROSS REFERENCE TO RELATED APPLICATION

This application claims benefit of U.S. patent application Ser. No.10/785,720, filed Feb. 24, 2004, which in turn claims benefit of U.S.provisional patent application No. 60/449,489, filed Feb. 24, 2003, bothof which are hereby incorporated by reference in their entireties.

GOVERNMENT INTEREST

This invention was made with government support under NIH Grant NumberDK57835, awarded by the National Institutes of Health. The U.S.government has certain rights in this invention.

FIELD OF INVENTION

The present invention relates to isolated polypeptides that selectivelyexpress in fat tissue, as well as methods for use of the polypeptides.

BACKGROUND OF THE INVENTION

Obesity affects a growing number of the U.S. population and often isclosely associated with insulin resistance, type 2 diabetes,cardiovascular disease, and dyslipidemia. Obesity itself is typically aheterogeneous condition, due to regional distribution of fat tissue.Central obesity generally refers to fat accumulation in omental orvisceral cavity, whereas peripheral obesity generally refers thesubcutaneous fat accumulation. Epidemiological studies have establishedthat central obesity is associated with a higher degree of risk thanperipheral obesity to the above-mentioned diseases, however, theunderlying mechanism(s) are generally not well understood. Presumably,distinctive biological properties of omental fat, in addition to itsunique anatomical location, contribute to the increased pathogenicity ofcentral obesity. It has been discovered that an excess of cortisol cancause central obesity and that treatment of HIV patients with proteaseinhibitor can lead to accumulation of omental fat accumulation butdepletion of subcutaneous fat. In vitro studies have also demonstratedthat abdominal visceral fat pads can be relatively resistant to theanti-lipolytic effect of insulin and susceptible to the lipolytic effectof catecholamine. At a molecular level, omental fat has been shown tohave increased gene expression or secretion of interleukin 6,plasminogen activator inhibitor (PAI-1), and angiotensinogen, comparedto subcutaneous fat. These observations indicate the existence ofbiological difference between omental and subcutaneous fat depots.

Adipose, i.e., fat, tissue plays a critical role in the pathogenesis ofobesity and its associated diseases, but the molecular mechanisms forthese associations generally remain unclear. Adipose tissue is generallyrecognized as an important endocrine organ that communicates activelywith the central nervous system and other peripheral tissues through therelease of a variety of bioactive factors that regulate glucose andlipid homeostasis. These factors, collectively known as adipocytokines,include leptin, tumor-necrosis factor α (TNFα), plasminogen activatorinhibitor-1 (PAI-1), adiponectin/ACRP30/adipoQ, and resistin.Adipocytokines have been demonstrated to play a key role in thepathogenesis of obesity and its associated diseases. Nevertheless,current knowledge of known genes generally cannot fully explain thepathophysiology of obesity, and effective treatment for these diseasesis still lacking.

Adipose tissue also plays a central role in energy homeostasis. Itsprimary function is to store and mobilize energy in the form oftriglycerides in response to caloric excess and deprivation,respectively. Adipose tissue can be divided into white adipose tissue(WAT) and brown adipose tissue (BAT). WAT and BAT are different inmorphology and biological function; WAT is monocular and mainlyfunctions in storing triglyceride, while BAT is multilocular, rich inmitochondria, and designed to burn energy. BAT is mainly present inhibernating animals, rodents and new-born humans, indicating evolutionaladaptation of adipose tissue to environment. Because the significance ofthe BAT to adult physiology is relatively not clear, references hereinto adipose tissue or fat cells generally refer to WAT. Central obesitygenerally refers to intra-abdominal fat accumulation in visceral oromental adipose tissues, although, strictly speaking, omental fat is asubset of visceral fat.

Obesity is due to the excess accumulation of triglyceride inintra-abdominal (omental) and subcutaneous adipose tissue. Studies ofanimal models have provided some mechanistic understandings of obesity,likely a communication disorder between the central nervous system andthe peripheral tissues, particularly adipose tissue. In mice, certainsingle gene mutations, such as db/db (leptin receptor mutation) andA^(y) (the agouti protein is homologous to melanocyte stimulatinghormone), can cause obesity, suggesting that a defect involving thecentral nervous system is the cause for the obesity in these animals.Leptin mutations also can cause obesity in mice (ob/ob), and in humans.However, obesity caused by single gene mutations in humans is rare, andobesity in the general population is thought to be of polygenic origin.

The association of type 2 diabetes with obesity has been observed for along time. Evidence from epidemiological, clinical and experimentalstudies has demonstrated that obesity is generally the greatest riskfactor for insulin resistance and type 2 diabetes and, moreover,visceral obesity is associated with a higher degree of risk thanperipheral obesity. The mechanism for the close association is generallynot well understood, but it is generally accepted that an excess of fatleads to increasing insulin resistance and/or impaired glucose disposal,which can predispose someone to type 2 diabetes. The pancreas, liver,muscle, fat tissue, and central nervous system are the principal organsinvolved in regulating glucose and fat metabolism and are likely toparticipate in the pathogenesis of obesity and type 2 diabetes. However,recent experimental studies indicate that fat tissue can play arelatively major role in the etiology of type 2 diabetes. For example,surgical excision of visceral fat tissue in the rat has been shown toincrease insulin sensitivity suggesting that excess fat is a causativefactor for type 2 diabetes. In addition, lipodystrophic patients andfat-depleted mice have developed hyperinsulinemia and type 2 diabetes,and surgical implantation of adipose tissue reverses the diabeticphenotype. Also, adipocyte size may be a determinant of body insulinsensitivity, as it has been proposed that small adipocytes conferinsulin sensitivity while large ones result in insulin resistance. Theseand other studies strongly support the premise that adipose tissue canplay a central role in the regulation of insulin sensitivity, and in thepathogenesis of type 2 diabetes.

As discussed above, fat cells generally play an active role in energystorage, fatty acid metabolism and glucose homeostasis. To perform thisspecialized function, the adipocyte expresses a special subset of genesto communicate with the central nervous system and peripheral tissues,and to respond to various neuronal, metabolic and hormonal signals. Theadipocyte secretes a number of bioactive substances, collectively knownas adipocytokines, such as, for example, leptin, TNFα, PAI-1,adiponectin, and resistin. These adipocytokines function as endocrine,paracrine, and autocrine factors, and have been implicated in obesityand its associated diseases. Some of these adipocytokines are discussedbriefly below.

Leptin is a hormone secreted from fat tissue into the circulation thatacts to reduce food intake and increase energy expenditure mainlythrough binding to leptin receptors in the hypothalamus. Leptinsecretion is regulated by the energy supply; starvation decreases itsexpression and secretion, while overfeeding or increased adiposityinduces leptin expression. Leptin is therefore a key molecule linkingthis adipose tissue to the central nervous system and regulating energyhomeostasis.

Tumor necrosis factor-alpha (TNFα) is a cytokine produced not only byinflammatory cells but also by adipocytes. TNFα expression has beenshown to be elevated in the fat tissue of obese animals and humans. TNFαappears to induce insulin resistance by interfering directly and/orindirectly with insulin signaling pathways in an autocrine or paracrinefashion. The absence of TNFα results in significantly improved insulinsensitivity in obese mice, the mice lacking TNFα receptors appearsprotected against diabetes to a certain degree, implying that theremight be a yet uncharacterized pathway involved in TNFα-induced insulinresistance.

Plasminogen activator inhibitor-1 (PAI-1) is a key pathogenic factor forthrombotic vascular disease. Plasma PAI-1 levels are closely correlatedwith visceral fat, and gene expression is highly elevated in visceralfat during the development of obesity. TNFα has been shown to induceadipose PAI-1 expression, providing a possible explanation for theassociation of obesity with cardiovascular disease.

Adiponectin is a hormone secreted exclusively from adipose tissue and isalso referred to as ACRP30, AdipoQ, apMI, or GBP28. Adiponectin has beendemonstrated to have promising activities potentially for the treatmentof obesity and diabetes. Its expression is reduced in the states ofobesity and type 2 diabetes, and its replenishment improves insulinsensitivity and prevents diet-induced obesity in rodents, probably byincreasing fat oxidation and decreasing triglyceride content in muscleand liver. This effect can result from increased expression of moleculesinvolved in both fatty-acid combustion and energy dissipation in muscle.The mechanisms for these actions are generally not clear. Adiponectinconsists of collagenous repeats and a globular domain homologous tocomplement C1q, and shares structural similarity to TNFα. Interestingly,PPARγ induces, whereas TNFα suppresses, the expression and secretion ofadiponectin, suggesting that adiponectin may be a target moleculerelaying insulin sensitivity.

Resistin is a hormone typically isolated from differentiated 3T3-L1adipocytes by screening for genes regulated by the PPARγ agonistrosiglitazone. During adipocyte differentiation, resistin isincreasingly expressed but is suppressed by treatment withrosiglitazone. Moreover, ob/ob mice secrete increased amounts ofresistin, and recombinant resistin induces insulin resistance. Resistinhas therefore been proposed to be a link between obesity and insulinresistance. However, conflicting results have been reported, in whichresistin expression was reduced in several obese animal models and wasinduced by PPARγ agonists. In addition, unlike the high expression ofmouse resistin in adipose tissue, the expression of the humancounterpart is very low.

There are additional adipocyte-specific/abundant genes, such as isadipsin and angiotensin, acylation stimulating protein, PGAR, andinterleukin-6, whose functions in obesity and type 2 diabetes aregenerally less understood. Nevertheless, the discovery of a myriad ofadipose secreted factors has generally established adipose tissue as anendocrine organ. The dysregulation of adipose tissues autocrine,paracrine and endocrine function is likely to disturb energy homeostasisand lead to obesity, type 2 diabetes, dyslipidemia and hypertension.

Abdominal fat is generally more pathogenic than subcutaneous fat. Anobvious explanation for this may simply relate to its anatomicallocation. Visceral adipose tissue drains via the portal venous system,such that liver is fully exposed to and functionally affected bybioactive substances released from this depot. In addition, differencesin physiology, biochemistry and gene expression have been observedbetween omental and subcutaneous fat tissues. Abdominal obesity ispredominant in males whereas subcutaneous fat mass is mostly involved infemale obesity, indicating that sex hormones may play a role in thesedifferences. Moreover, an excess of cortisol is known to cause centralobesity. Finally, a selective increase in visceral fat is a commonfeature of aging. It has been suggested that these two adipose tissuedepots differ in important ways. Omental adipose fat is moremetabolically active with respect to lipolysis and lipogenesis. Comparedto subcutaneous fat, abdominal fat pads have greater secretion ofinterleukin 6, plasminogen activator inhibitor (PAI-1), angiotensinogen,and the rate of apoptosis is greater. In contrast, leptin expression ishigher in subcutaneous fat tissue than omental fat tissue. Yet, whetherthese changes discussed above can explain features of insulin resistancesyndrome generally remains unclear. Because the pathophysiological basisof this syndrome is likely to be complex, several genes/gene productsand pathways may participate in the disease process.

Insulin signaling is a complex and coordinated process involving proteinmodification, translocation, and compartmentalization. Insulin action isinitiated through binding of insulin to the α subunit of insulinreceptor (IR), which activates the beta subunit intrinsic receptortyrosine kinase, resulting in autophosphorylation of insulin receptor βsubunit and tyrosine phosphorylation of intracellular target proteinssuch as IR substrates (IRS-1-4) and Shc, Cb1, Gab-1. Three majorsignaling pathways are initiated by these intracellular targets: 1)IRS/PI 3-kinase/Akt; 2) CAP/Cb1; and 3) Shc(or Gab)/Ras/MAP kinase.

In the first major pathway, tyrosine-phosphorylated IRS-1 or IRS-2 bindsto src-homology 2 domains of intracellular proteins, including p85, aregulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase). Theinteraction of IRS and p85 subunits results in the activation of thep110 catalytic subunit of PI 3-kinase, which raises phosphatidylinositol3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (PIP3)levels. These second messengers activate phosphoinositide-dependentkinase-1 (PDK-1) to phosphorylate and hence activate Akt (also calledprotein kinase B) and atypical PKC isozymes.

In the second major pathway, c-Cbl-associated protein (CAP) recruitsc-Cbl to the insulin receptor where it is phosphorylated. This proteincomplex subsequently localizes to lipid raft domains of the plasmamembrane called caveola. The SH2-containing adapter protein CRKII andC3G, a guanine nucleotide exchange factor, are then targeted tophosphorylated c-Cbl at the lipid raft. C3G may activate TC 10, aG-protein of the rho family, which is expressed in adipose and muscletissue. The IRS/PI 3-kinase/Akt and CAP/Cbl pathways are generallybelieved to function in concert to upregulate glucose transport inresponse to insulin.

The third major pathway involves the activation of the p42/44 MAP kinase(mitogen activated protein kinase) cascade. Insulin receptorphosphorylation of both Shc and Gab-1 adaptor proteins leads to Rasactivation of multiple kinases resulting in activation of MAP kinase(Erk1 and 2). This pathway is more involved in the mitogenic function ofinsulin.

Many other factors interact with and modify the efficiency of insulinsignaling in a positive or negative manner, which include proteinkinases, e.g., AMP-activated kinase, protein kinase C, and IKKβ,phosphatases, e.g., PTP1B, SHIP2, PTEN, and modulators of IR activity,e.g., PC-1.

There is a need for methods of detecting and treating diseases of orrelating to adipose tissue and glucose metabolism, such as obesity andtype 2 diabetes.

SUMMARY OF THE INVENTION

An object of this invention is to provide a method for modifying insulinaction and/or glucose metabolism in an animal, such as, for example, ahuman.

Another object of this invention is to provide a method for inducingglucose uptake by animal adipocytes.

An additional object of this invention is to provide a therapeutictreatment for diseases of or related to metabolism and/or adiposetissues.

Yet another object of this invention is to provide a method ofdiagnosing or detecting a disease or condition involving animal tissuethat contains, uses, or expresses omentin polypeptide in an animalsuspected of having the disease or condition.

Yet another object of this invention is to provide a method and/or adiagnostic kit for detecting a polypeptide specific to particular fattissues, such as omental fat tissue, in bodily fluids of an animal.

One object of the invention can be attained, at least in part, through amethod of modifying at least one of insulin action and glucosemetabolism in an animal. The method includes modifying the amount ofomentin polypeptide in the animal. The amount of active omentinpolypeptide can be increased in the animal, such as by administeringomentin polypeptide to the animal, or decreased in the animal byinterfering with the metabolic function of at least a portion of theomentin polypeptide in the animal. In one embodiment of this invention,the amount of omentin polypeptide in the animal is first determinedbefore any modification.

The invention further comprehends a method of detecting omentinpolypeptide in bodily fluids of an animal. The method includescontacting a sample of the bodily fluids with at least one antibody thatspecifically binds to the omentin polypeptide. The antibody bound to theomentin polypeptide in the sample is then detected.

The invention still further comprehends a method of diagnosing ordetecting a disease or condition involving animal tissue that contains,uses, or expresses omentin polypeptide in an animal suspected of havingthe disease or condition. The method includes first contacting a sampleof bodily fluid from the animal with a plurality of antibodies adaptedto specifically bind omentin polypeptide. The antibody bound to omentinpolypeptide in the sample is detected and an amount of omentinpolypeptide in the bodily fluid is measured. The amount of omentinpolypeptide is compared to a control to diagnose or detect the diseaseor condition.

The invention still further comprehends a diagnostic kit for use indiagnosing damage, a condition, or disease in tissue containing orexpressing omentin polypeptide. The diagnostic kit includes a measurerof an amount of omentin polypeptide in a sample of bodily fluids and anindicator for determining if a measurement taken by the measurer is in apredetermined range associated with damage, a condition, or disease inthe tissue.

The invention still further comprehends a method of inducing glucoseuptake by animal cells, tissues, and/or organs, such as, for example,adipocytes or adipose tissue. The method includes administering anomentin polypeptide to at least one of the animal and the adipocytes.The administered omentin polypeptide can enhance insulin-mediatedglucose transport in the adipocytes and/or activate, either directly orindirectly, the polypeptide kinase Akt/PKB, also referred to herein as“Akt kinase.”

As discussed above, obesity is a heterogeneous condition and can bedivided into central (omental or visceral) obesity and peripheral(subcutaneous) obesity, based on the location of fat accumulation.Central obesity is more closely associated with insulin resistance, type2 diabetes and cardiovascular disease than peripheral obesity, but theunderlying mechanisms are generally not known, presumably due to thebiological and anatomical difference between the two fat depots. In thisinvention, fat depot-specific secretory factors have been identified,such as, for example, proteins having the amino acid sequences of SEQ IDNO:1 and SEQ ID NO:3, as well as variants thereof, generally referred toherein as “omentin” or “omentin polypeptides.” Omentin polypeptides areexpressed in the stromal vascular cells derived from omental, butgenerally not subcutaneous, adipose tissue. Omentin polypeptides enhanceinsulin-mediated glucose uptake by adipocytes in vitro and improveglucose disposal in vivo. Furthermore, omentin polypeptides activate Aktkinase, both alone and synergistically with insulin. Omentinpolypeptides are also detectable in human blood.

Insulin is a pleiotropic hormone with a broad spectrum of biologicalfunctions. In particular, it plays a vital role in regulating glucose,lipid and protein metabolism. The maintenance of glucose homeostasisdepends on a precise balance between the release of insulin from thepancreas, glucose production from the liver, and insulin-stimulatedglucose transport by muscle and adipose tissue. A number of biologicalfactors can alter insulin sensitivity via different mechanisms.Adipocytokines have been demonstrated to either positively, e.g., leptinand adiponectin, or negatively, e.g., TNFα, modulate insulinsensitivity. Omentin polypeptides can enhance insulin-stimulated glucosein 3T3-L1 adipocytes.

As discussed above, adipocytokines generally play a role in regulatingenergy metabolism and insulin action. Omentin polypeptides arephysiological regulators in this regard as well. Omentin polypeptidesare secretory factors from stromal vascular cells in the omental depotand are detectable in human blood. Omentin polypeptides are biologicallyactive in enhancing insulin action in vitro and in vivo. Therefore, theomentin polypeptide is an adipokine that can regulate the adiposebiology in a depot-dependent manner.

In identifying and purifying omentin polypeptides, 10,437 expressedsequence tags (EST) from a human omental fat library were sequenced.Bioinformatics analysis revealed that one frequently sequenced EST was apotential secretory factor and Northern analyses revealed that this ESTwas expressed in omental, but not in subcutaneous, adipose tissue bothin humans and Rhesus monkeys. In one embodiment of this invention, theomentin polypeptide is 313 amino acids in length. Omentin polypeptidesare secreted proteins when expressed in mammalian cells. In addition,omentin polypeptide can be detected in human blood by Western blotting.Immunofluorescence microscopy demonstrates that omentin polypeptides aregenerally expressed by stromal vascular cells in omental fat.

In one embodiment of this invention, omentin polypeptides enhanceinsulin-mediated glucose transport in 3T3-L1 adipocytes. In anotherembodiment of this invention, omentin polypeptides activate the proteinkinase Akt/PKB, both in the presence and absence of insulin. Omentinpolypeptides also stimulate insulin-mediated glucose transport and Aktphosphorylation in vitro. This activity is believed to explain, at leastin part, why the omental adipose tissue continues to accumulate fatdespite systemic insulin resistance in obesity. Without intending to bebound by theory, it is believed that omentin polypeptides sensitizeadipocytes to insulin by activating components of the insulin signalingpathway and/or inhibiting negative regulators of the pathway.Furthermore, omentin polypeptide levels can be correlated with visceralfat, increased glucose disposal, and increased prevalence of type 2diabetes.

In one embodiment of this invention, the gene encoding the omentinpolypeptide provides a positional candidate gene for type 2 diabetesbecause this gene localizes to a region on chromosome 1q22 that has beenlinked to type 2 diabetes susceptibility in the Old Order Amish and inat least four other populations independently.

As used herein, the terms “omentin.” “omentin protein,” or “omentinpolypeptide” generally refer to a polypeptide having an amino acidsequence with at least 70% identity, preferably at least 80% identity,more preferably at least 90% identity, and desirably at least 95%identity, to the amino acid sequence of either SEQ ID NO: 1 or SEQ IDNO:3.

As used herein, the term “animal” is intended to include humans.

Other objects and advantages will be apparent to those skilled in theart from the following detailed description taken in conjunction withthe appended claims.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are blots from Northern analyses. FIG. 1A is a multipletissue Northern analysis blot from a human. FIG. 1B is a Northern blotof adipose tissues from omental and subcutaneous fat depots of fiveindividual humans.

FIGS. 2A and 2B are blots from Northern analyses. FIG. 2A is a Northernblot of paired omental (O) adipose tissue and inguinal subcutaneous (S)adipose tissue from five individual Rhesus monkeys. FIG. 2B is aNorthern blot that shows regional differences in omentin expression in asingle Rhesus monkey.

FIG. 3 is a schematic structure of the omentin polypeptide of SEQ ID NO:1.

FIG. 4A is a structural representation of a His (6)-tagged omentinpolypeptide cDNA in a PET-28 plasmid.

FIG. 4B is a Coomassie Blue stain of bacterial cell lysates expressingrecombinant human omentin polypeptide.

FIG. 4C is a Western blot of recombinant human omentin polypeptide.

FIG. 5 is a chromosome map showing the location of the omentin gene byits peak linkage to diabetes phenotypes in chromosome 1q21-q23.

FIG. 6 is an immunoblot of proteins from conditioned medium and celllysate of example HEK-293 cells stably transfected with an omentin-Fvector (+).

FIG. 7 is a Western blot of three plasma samples immunoprecipitated withomentin antibodies (“Om”) and pre-immune antibodies (“Cont”).

FIGS. 8A-D are photographs of immunofluorescent stained fat tissue. FIG.8A is omental tissue treated with pre-immune antibody. FIG. 8B isomental tissue treated with omentin antibody. FIG. 8C is subcutaneoustissue treated with omentin antibody. FIG. 8D is omental tissue treatedwith omentin antibody shown at a greater magnification (200×) than thesample of FIG. 8B (100×).

FIG. 9 is a graphical representation of the results of a glucosetransport assay.

FIG. 10 is a graphical representation of the results of a glucose totransport assay.

FIG. 11 is a Western blot showing Akt kinase phosphorylation by acuteand chronic exposure to omentin-F.

FIG. 12 is a graphical representation of the results of an in vivoglucose transport test.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention relates to polypeptides, referred to hereingenerally as omentin or omentin polypeptide, selectively expressed inhuman and other animal omental fat depots, and generally not insubcutaneous fat depots, as well as methods of use of the polypeptides.

In one embodiment of the invention, the omentin polypeptide includes anamino acid sequence of SEQ ID NO:1. The nucleotide sequence for thecomplimentary DNA (cDNA) of SEQ ID NO:1 is SEQ ID NO:2. The presentinvention also includes variants of the polypeptide of SEQ ID NO:1,i.e., polypeptides that vary by conservative amino acid substitutions,whereby a residue is substituted by another with like characteristics.

In one embodiment of this invention, the variant polypeptides for use inthe methods of this invention have at least 70% identity, preferably atleast 80% identity, more preferably at least 85% identity, morepreferably at least 90% identity, and desirably at least 95% identity,to the amino acid sequence of SEQ ID NO:1, over the entire length of SEQID NO:1. One preferred homolog of this invention and usable in themethods of this invention is a polypeptide including the amino acidsequence of SEQ ID NO:3. The polypeptide of SEQ ID NO:3 has about 86%similarity and about 84% identity with the polypeptide of SEQ ID NO:1.The nucleotide sequence for the complimentary DNA (cDNA) of SEQ ID NO:3is SEQ ID NO:4.

Polypeptides for use in the methods of this invention can be prepared inany suitable manner. Such polypeptides include isolated naturallyoccurring polypeptides, recombinantly produced polypeptides,synthetically produced polypeptides, and/or polypeptides produced by acombination of these methods. As will be appreciated by one skilled inthe art following the teachings herein provided, various means forpreparing such polypeptides are available in the art.

The omentin polypeptide of this invention was isolated and purified fromadipose, i.e., fat, tissue. Messenger RNA encoding omentin polypeptideis relatively highly and selectively expressed in omental fat tissue.FIG. 1A shows the results of a multiple tissue Northern analysis blotfrom a human. As shown in the Northern blot of FIG. 1A, messenger RNAencoding omentin polypeptide is selectively expressed in omental fattissue, much less in lung and heart, and not at all in muscle, liver,kidney, breast, and brain. FIG. 1B is a Northern blot of adipose tissuesfrom omental and subcutaneous depots of five individual humans. As shownin the Northern blot of FIG. 1B, messenger RNA encoding omentinpolypeptide is relatively highly expressed in omental fat, but not insubcutaneous fat. In the Northern analysis represented in FIG. 1B, theblot was reprobed with leptin, showing that leptin is preferentiallyexpressed in subcutaneous fat, and generally not in omental fat, as isgenerally known in the art. For the Northern analyses resulting in FIGS.1A and 1B, adipose RNA was prepared with TRIZOL, available fromInvitrogen, Carlsbad, Calif., from human tissue. The other RNA sampleswere obtained from Clontech, Palo Alto, Calif. The Northern analysis wasperformed by loading 15 μg of total RNA per lane. The human omentinprobe, corresponding to bases 263 to 1270 of AY549722 in GenBank, wasrandom-labeled, available from Stratagene, La Jolla, Calif., with³²P-dCTP. Hybridization was carried out at 65° C. in Rapid-hyb buffer,available from Amersham Biosciences, Piscataway, N.J., and blots werewashed twice with 0.5×SSC/1% SDS at 65° C. The RNA loadings wererevealed by ethidium bromide staining.

To eliminate the possibility of individual variations in gene expressionamong different subjects, Northern analysis was also conducted usingsamples from Rhesus monkeys in the manner described above. FIG. 2A is aNorthern blot of paired omental (O) adipose tissue and inguinalsubcutaneous (S) adipose tissue from five individual Rhesus monkeys. Asshown in the Northern blot of FIG. 2A, messenger RNA encoding omentinpolypeptide was predominantly expressed in omental fat compared tosubcutaneous fat from the same animals. FIG. 2B is a Northern blot thatshows regional differences in omentin expression in a single Rhesusmonkey. To help rule out concern of potential contamination from omentalstructures other than omental fat, adipose tissues from differentregions were obtained from a single monkey for Northern analysis. Asshown in FIG. 2B, omentin is expressed in intrathoracic as well as inomental fat tissue, but not in the subcutaneous fat tissues from neckand inguina, confirming the visceral pattern of omentin expression.

The omentin polypeptide of SEQ ID NO:1 contains 313 amino acids. Theestimated molecular weight is 35 kDa. FIG. 3 is a schematic structure ofthe omentin polypeptide of SEQ ID NO:1. The N-terminal portion (the endat amino acid 1) of omentin polypeptides contain a hydrophobic regionthat is typical of a signal sequence for protein secretion, followed bya region homologous to a fibrinogen-related domain (FBG). Such domainsare globular structures found in proteins, such as the β and γ chains offibrinogen, PGAR (PPARγ angiopoietin related), and tenasin. Full-lengthclones of omentin were obtained from EST sequencing of an omental fatcDNA library, Cat# HL-5028t, available from Clontech. The EST clone wasused as a template for PCR amplification of protein-coding regions withappropriate primers. The resultant PCR products were subcloned intoproper expression vectors and verified without mutation by sequencing.

In one embodiment of this invention, omentin polypeptide is expressedusing a His (6)-tagged omentin cDNA in a bacteria host cell as shown inFIG. 4A. Omentin polypeptide without the signal peptide was cloned intoa PET28 vector, available from Novagen, San Diego, Calif., under thecontrol of the T7 promoter. The target gene was introduced into Tuner(DE3) pLacI cells, expressed by IPTG induction, and purified with anickel column. The omentin-containing bacteria were grown with (+) orwithout (−) IPTG induction. Cell lysates, shown in the first two lanesof FIG. 4B, and purified protein, shown in lane 3 of FIG. 4C, wereanalyzed on 4-20% SDS-PAGE and stained with Coomassie Blue.

In one embodiment of this invention, antibodies are formed from theomentin polypeptide. As will be appreciated by one skilled in the artfollowing the teachings herein provided, various methods are availablein the art for forming antibodies. For antibody production, cDNAencoding amino acids 73-313 of SEQ ID NO:1 was amplified with primer1120 and 1121 using high-fidelity PCR system, available from BoehringerMannheim, Mannheim, Germany, and subcloned into a PET28 vector,available from Novagen, to create the plasmid G6329, which was verifiedby sequencing and transformed into Escherichia coli, available fromNovagen. The His(6)-tagged protein was over-expressed by IPTG induction(Yang R. Z., Blaileanu G., Hansen B. C., Shuldiner A. R., and Gong D.W., cDNA cloning, genomic structure, chromosomal mapping, and functionalexpression of a novel human alanine aminotransferase, Genomics, 2002March; 79(3):445-50, herein incorporated by reference in its entirety),and purified with Ni2+-NTA resin, available from QIAGEN Inc., Valencia,Calif., under denaturing condition using 8M urea for polyclonal antibodyproduction in rabbits and monoclonal antibody production by AnaSpec, SanJose, Calif.

A representative immunoblot utilizing antibodies obtained in this manneris shown in FIG. 4C. The indicated amount of recombinant omentinpolypeptide was blotted to a nitrocellulose membrane and probed withomentin antiserum or pre-immune serum, followed by a second antibody,such as alkaline phosphatase-labeled goat against rabbit IgG, stainedwith BCIP/NBT. A clear induction of a recombinant protein was observedat the expected size (about 35 kd) and purified to homogeneity. As shownin FIG. 4C, at about a 1 to 5,000 dilution factor, the antiserum reactswith recombinant omentin polypeptide in a dose-dependent manner, whereasthe pre-immune serum shows no staining.

The gene encoding omentin polypeptides is localized on a region of humanchromosomes generally known to be linked to type 2 diabetes. The omentingene consists of eight exons and localizes to chromosome 1q22 at 160.1cM, close to STS marker SHGC-31641. This region of human chromosomes hasbeen linked with type 2 diabetes. FIG. 5 shows, by the arrow, thelocation of the omentin gene relative to the peak linkage signal at1q21-q23, known to be linked to diabetes in the Old Order Amishpopulation. As will appreciated by one skilled in the art reviewing FIG.5, evidence for linkage in this region is stronger for the combinedtrait of diabetes and impaired glucose homeostasis (lod=2.4) than fordiabetes alone (lod=0.92). This region on chromosome 1 is known torepresent the most strongly replicated linkage peak for type 2 diabetes.Lod scores reported from other studies corresponding to this regionrange from 4.3 in Utah Mormons, to 3.0 in collection of French families,to 2.5 in Pima Indians.

As discussed above, omentin polypeptide is a secretory protein.Bacteria-expressed omentin polypeptide is generally insoluble andrenaturing efforts generally failed to recover the protein in a solublefraction. In one embodiment of the invention, to obtain the solubleprotein, a flag peptide sequence is tagged to the coding region ofomentin polypeptide at the carboxy terminus by polymerase chain reaction(PCR). The PCR product is subcloned into a mammalian expression vector,such as, for example, pcDNA3, available from Invitrogene Corporation,Carlsbad, Calif., driven by a CMV promoter. The resultant plasmid,referred to as omentin-F, is confirmed by sequencing and used totransiently transfect HEK-293T cells using Transfectamine Plus,available from Invitrogene Corporation. The cells are grown in DMEM with10% FBS. The culture medium was collected and cells are lysed 48 hoursafter transfection for immunoprecipitation. The fractions areimmunoprecipitated with anti-flag M2 affinity beads, available fromSigma-Aldrich, St. Louis, Mo., and blotted with omentin antibody. As aresult, omentin-F is detected in both culture media and cell lystatefrom the cells transfected with omentin-F plasmid, but not from emptyvector-transfected control cell. FIG. 6 is an immunoblot of proteinsfrom conditioned medium and cell lysate of example HEK-293 stabletransfected with an omentin-F vector (+) according to the abovedescribed method, or an empty vector (−). Culture medium, in the amountof about 5 milliliters, and one third of the cell lysates from a 10centimeter dish were immunoprecipitated with M2-Flag antibody beads. Theprecipitates were separated on 10% SDS-polyacrylamide gelelectrophoresis, immunoblotted with omentin antibodies, and detectedwith ECL, available from Amersham Biosciences.

Omentin polypeptide was detected in human plasma by immunoprecipitating3 milliliters of each of three individual plasmas. The three plasmasamples were immunoprecipitated with omentin antibodies (“Om”) orpre-immune antibodies (“Cont”). The precipitates were analyzed byWestern blotting using omentin antiserum or pre-immune serum. FIG. 7 isa Western blot showing the results of the analysis. The arrow indicatesthe specific band at the expected size (about 38 Kd) of omentin. Celllysates or immunoprecipitates were separated by SDS-PAGE using 4-20%polyacrylamide gels, available from Gradipore Inc., Hawthorne, N.Y.Following electrophoresis, proteins were transferred onto PVDF membranesand bound proteins were detected by blotting with primary antibody.Immunodetection was achieved by chemiluminescence using ECL, availablefrom Pierce Biotechnology, Rockford, Ill. For human fat explant testing,human biopsy adipose tissues were minced and cultured in 199M medium for4 hours. The cultured media were concentrated 30 times before beingsubjected for Western blot analysis with monoclonal anti-omentin 3GXX.For detection of circulating omentin in humans, human plasmas (3 ml,from the Blood Bank of the University of Maryland) were incubated with20 μl of omentin antiserum or pre-immune (control) and protein ASepharose beads at 4° C. for 4 hours. The beads were precipitated,washed and subjected to immunoblotting with anti-omentin polyclonalantibody.

Immunofluorescent staining, as shown in FIGS. 8A-D, demonstrates thatomentin polypeptide is actually expressed in vivo, and defines thecellular localization. Human adipose tissues was cryosectioned (tissuesamples were about 20 μm thick), fixed with 3% paraformaldehyde andpermeabilized with 0.5% cold Triton X-100. The tissue slides were thenincubated with polyclonal anti-omentin or pre-immune IgG (2.5 mg/ml inPBS) at 1:1000 dilution, washed and re-incubated with a second antibody,goat anti-rabbit IgG-Alexa 568, available from Molecular Probes, Eugene,Oreg. The slides were counterstained with nuclei (DAPI). FIGS. 8A-D showthe immunofluorescent staining of the samples. As shown in FIGS. 8A-D,the omentin staining illustrates the unique structure of the adipocyte,with a large central triglyceride storage droplet (round empty circle)and a thin rim of cytoplasm and cell membrane. The fluorescence (lighterportions) is clearly visible in the omentin antibody-stained tissue andmuch less evident in tissue stained with the pre-immune antibodies. Inaddition, smaller cells appear to be more intensively stained than thelarge adipocytes. It is believed that these smaller cells representstromal vascular cells. FIGS. 8A-D also illustrate that omental, but notsubcutaneous, adipose tissue contains immunoreactive omentinpolypeptides by immunofluorescence staining. Again it is shown thatomentin polypeptides are selectively expressed in the omental adiposetissue.

The selective expression of omentin polypeptide in omental fat tissueand the metabolic effect of omentin polypeptide allow for the use ofomentin polypeptides in diagnosing and treating injuries or diseases ofor related to adipose tissue. In one embodiment of this invention, amethod of modifying at least one of insulin action and glucosemetabolism in an animal includes determining an amount of omentinpolypeptide in the animal. The amount of omentin polypeptide in theanimal can be modified as needed to obtain a desired result. The methodsof this invention can include one or more of any of the omentinpolypeptides disclosed herein, including the polypeptides having anamino acid sequence of one of SEQ ID NO:1 and SEQ ID NO:3, andhomologous variants thereof.

In one embodiment of the invention, modifying the amount of the omentinpolypeptide in the animal includes increasing the amount of omentinpolypeptide in the animal. The amount of omentin polypeptide can beincreased by administering omentin polypeptide to the animal. In anotherembodiment of the invention, the amount of omentin polypeptide in theanimal can be modified by decreasing or interfering with the omentinpolypeptide in the animal. The metabolic function of at least a portionof the omentin polypeptide in the animal can be interfered with byintroducing an amount of a molecule that regulates signaling orfunctioning of the omentin polypeptide. As will be appreciated by oneskilled in the art following the teachings herein provided, in oneembodiment of this invention, the molecule is any molecule thatinterferes with receptor binding of the omentin polypeptide. Examples ofsuch molecules, include, for example, antibodies or other proteins orpolypeptides. By increasing or decreasing the effective amount ofomentin, the method of this invention can be used to therapeuticallytreat an undesirable condition of tissue or a disease, such as obesityand type 2 diabetes, such as by modifying or manipulating glucosemetabolism.

In another embodiment of this invention, the omentin polypeptide is usedin a method of inducing glucose uptake by at least one of animal cells,tissues, and organs, such as, for example adipose tissue, adipocytes,the liver and cells and tissues thereof, the brain and cells and tissuesthereof, muscles and cells and tissues thereof, the kidney and cells andtissues thereof. The method includes administering an omentinpolypeptide to either the animal in vivo, or adipocytes in vitro. Theomentin polypeptide can include one or more of any of the variants ofomentin polypeptide described above. In one embodiment of the invention,the administered omentin polypeptide enhances insulin-mediated glucosetransport in the adipocytes, thereby inducing glucose uptake by animaladipocytes. In another embodiment the administered omentin polypeptideactivates, either directly or indirectly, the polypeptide kinaseAkt/PKB, thereby inducing glucose uptake by animal adipocytes.

In one embodiment of this invention, omentin polypeptide can be used toincrease insulin-stimulated 2-deoxyglucose transport. To demonstrateomentin polypeptide effect on insulin-stimulated 2-deoxyglucosetransport, a cassette of pIRES2-hrGFP, available from Stratagene, wassubcloned in pcDNA3 backbone via appropriate shuttle vectors, therebycreating the plasmid G6422. The plasmid was transfected into mammalianHEK-293T cells and the top 10% of cells with highest fluorescence,sorted by fluorescence-activated cell sorting (FACS), were collected andcultured. The cells were grown in 10% FBS DMEM medium, available fromInvitrogen, Carlsbad, Calif., to 80% confluency and then in serum-freeDMEM for 5 days. The conditioned medium was subjected to SuperQ anionion exchange chromatography, available from Tosoh Biosciences,Montgomeryville, Pa., and the omentin-containing fractions were pooledfor galactose-affinity chromatography, available from PierceBiotechnology, Inc. The protein was eluted with a buffer containing 10mM Tris-1 mM EDTA (at about pH 8) and used for analysis and the elutionbuffer was used as control. Typically, 3 liters of cell culture mediayield about 100 μg omentin-F. The secretion of omentin into the mediumwas verified by Western blotting with omentin antibody. Cells with thehighest omentin expression were selected and then cultured first ingrowth medium (DMEM/10% FBS) and then replaced with serum-free medium(DMEM only) for 48 hours.

The conditioned media were collected and concentrated 100 times withCENTRICON-80 filters (Millipore, 10 kDa filter) for a glucose transportassay using differentiated 3T3-L1 adipocytes. The glucose transportassay was conducted according to Kashiwagi A., Verso M. A., Andrews J.,Vasquez B., Reaven G., Foley J. E., In vitro insulin resistance of humanadipocytes isolated from subjects with noninsulin-dependent diabetesmellitus, J Clin Invest. 1983 October; 72(4): 1246-54, hereinincorporated by reference in its entirety. Briefly stated, humanadipocytes were isolated by collegenase digestion and centrifugation.The cells were incubated with omentin (500 ng/ml) for 30 minutes andstimulated by insulin (60 nM) for 5 min. The glucose transport wasdetermined by measuring the uptake of a trace concentration of glucose.The results of the glucose transport assay are represented in FIG. 9. Asshown in FIG. 9, omentin polypeptide stimulates the glucose uptake atall insulin concentrations tested. The omentin polypeptide exhibited itslargest effect (1.5 to 2-fold increase) on insulin-stimulation between 2and 20 nM.

To further demonstrate the affect of omentin polypeptide oninsulin-signaling, adipocytes from human fat tissue were isolated andexposed to insulin with or without pretreatment of omentin polypeptide.FIG. 10 is a graphical representation of the results of the analysis. Asshown in FIG. 10, insulin stimulated the uptake of [14C]2-deoxyglucoseabout 50%. An additional 20-30% more glucose uptake was observed withthe pretreatment of omentin (30 minutes before insulin).

Omentin polypeptides stimulate Akt kinase acutely and synergisticallywith insulin. Akt kinase is activated by insulin and other growthfactors, and is a central molecule mediating cell growth, proliferation,and apoptosis. Activation of Akt kinase requires phosphorylation atthreonine (T308) and serine (S473) residues. To demonstrate omentinpolypeptide affect on Akt phosphorylation, isolated adipocytes wereincubated with omentin polypeptide for various times and then treatedwith or without insulin (60 nM). The reaction was stopped by addinglysis buffer containing 2% SDS, 62.5 mM Tris-HCl, pH 6.8 at 90° C.,followed by sonication for 15 seconds. The resultant lysates weresubjected to immunoblotting analysis with a mixture of twophosphor-specific antibodies against the phosphorylated Akt or withgeneral Akt antibody for total Akt in the lysates. Omentin polypeptideactivated Akt kinase acutely, within about 15 minutes, by a treatmentwith serum-free conditioned medium (about 200 ng/ml omentin). Additionalactivation of Akt kinase by omentin polypeptide was observed in thepresence of insulin, indicating a synergistic effect between the twofactors. FIG. 11 shows Akt kinase phosphorylation by acute and chronicexposure to omentin-F. Adipocytes (3T3-L1) were exposed to omentin-F for15 minutes and 2 hours, followed by stimulation with 0, 0.5, or 2 nMinsulin for five minutes. Cell lysates were immunoblotted with Aktantibodies for Western blotting. As shown in the Western blot of FIG.11, after a two-hour exposure to omentin polypeptide, when maximaleffects on glucose transport were observed, activation of Akt kinase waslower but still elevated over the control (“None”).

To demonstrate the impact of omentin polypeptide on glucose uptake inadipocytes in vivo, mice were administered intraperitoneally withomentin polypeptide (1 μg/g body weight) or control vehicle (PBS) 30minutes prior to administering glucose. To obtain large quantity ofomentin for animal study, the signal peptide of omentin (16 amino acidsat the amino-terminal) was replaced with a His (6) tag and the fusionprotein was expressed in bacteria using a similar approach as describedin Yang et al., previously incorporated by reference in its entirety.The bacterially-expressed omentin was an inclusion body and insoluble.The inclusion body was purified and was dissolved in CAPS, availablefrom Novagen, according to the manufacture's protocol. The solublizedprotein was dialyzed and further purified by gel filtration with Hload16/60 Superdex 200, available from Amersham Biosciences, intohomogeneity. C57/BL mice were obtained from Jackson Laboratory, BarHarbor, Me. The mice were housed on a 12-hour light/dark cycle andallowed free access to standard mouse food and water. The mice were usedfor testing were at 7-10 weeks of age. For testing, the mice fastedovernight and received an intraperitoneal injection of omentin (1 μg/gbody weight) or only vehicle, as a control, 30 min before anintraperitoneal injection of glucose load at 2 mg/g body weight. Bloodsamples were obtained via tail veins at the indicated times before andafter the glucose injection. Blood glucose concentrations were measuredwith an ACCU-CHEK blood glucometer, available from Roche Diagnostics,Indianapolis, Ind.

FIG. 12 is a graphical representation of the results of the analysis.Upon intraperitoneal glucose injection, blood glucose levels in the micerose rapidly from roughly 100 to 400 mg/dl within 15 minutes in bothgroups. However, the glucose levels in the control group (dotted line)peaked at about 30 minutes and decreased thereafter. In contrast, theglucose levels in omentin-administered group (solid line) began to dropfrom 30 minutes, and were lower than the control group over the two hourperiod. This result demonstrates that omentin polypeptide improvesglucose disposal in vivo.

Omentin polypeptide thus enhances insulin action by stimulatinginsulin-mediated glucose uptake or disposal and improves glucosetolerance.

The invention additionally relates to a method of diagnosing ordetecting a disease or condition involving animal tissue that contains,metabolically uses, or expresses omentin polypeptide in an animalsuspected of having the disease or condition. In one embodiment of thisinvention, a first step of the method includes contacting a sample ofbodily fluid from the animal with a plurality of antibodies adapted tospecifically bind omentin polypeptide. The antibodies bound to omentinpolypeptides in the sample can then be detected and measured. As will beappreciated from the discussions above, levels of omentin polypeptidevary within different humans or other animals. By comparing the measuredamount to a control, the omentin polypeptide can be used to diagnose ordetect a disease or condition, particularly those of or related toparticular tissues, such as, for example, liver tissue, brain tissue,muscle tissue, adipose tissue, and kidney tissue. The bodily fluid fromthe animal used for testing can include, without limitation, blood,serum, lymph, urine, sweat, mucus, sputum, saliva, semen, spinal fluid,interstitial fluid, synovial fluid, cerebrospinal fluid, gingival fluid,vaginal fluid, and pleural fluid.

As omentin polypeptides are secreted and measurable in a human or otheranimal, another aspect of this invention includes diagnostic tests andkits for detecting and/or measuring the amount or level of omentinpolypeptide in a human or other animal. The omentin polypeptide level ina human or other animal can be used for testing for particular diseases,such as, for example, obesity and type 2 diabetes, or a susceptibilityto such diseases.

One embodiment of this invention includes a method of detecting omentinpolypeptide, or the amount thereof, in bodily fluids of an animal. Inone particularly preferred embodiment of this invention, the methodincludes first contacting a sample of the bodily fluids with at leastone antibody that specifically binds to the omentin polypeptide. Then,the antibody bound to the omentin polypeptide in the sample is detectedand, optionally, measured, such as by means available and know in theart. The bodily fluid to be tested can be any bodily fluid includingblood, serum, lymph, urine, sweat, mucus, sputum, saliva, semen, spinalfluid, interstitial fluid, synovial fluid, cerebrospinal fluid, gingivalfluid, vaginal fluid, and pleural fluid. The omentin polypeptide can beany omentin polypeptide disclosed herein, including variants thereof notparticularly disclosed.

The invention additionally includes a diagnostic kit for use indiagnosing damage or disease in tissue containing or expressing omentinpolypeptide. In one particularly preferred embodiment of this invention,the diagnostic kit includes a measurer of an amount of omentinpolypeptide in a sample of bodily fluids. The measurer, for example, caninclude a biologic assay, an antibody-based assay, an enzyme linkedimmunosorbent assay, a Western blot, a rapid immunoassay, and aradioimmunoassay. The diagnostic kit also includes an indicator, suchas, for example, a control or a list of predetermined values forcomparison, for determining if a measurement taken by the measurer is ina predetermined range associated with damage or disease in the tissue.

Thus, the invention provides isolated polypeptides that selectivelyexpress in fat tissue as well as methods for use of the polypeptide. Thepolypeptides can be used therapeutically to regulate or modify glucosemetabolism, thereby providing a therapeutic agent for diseases, or in adiagnostic test or kit for detecting diseases.

The invention illustratively disclosed herein suitably may be practicedin the absence of any element, part, step, component, or ingredientwhich is not specifically disclosed herein.

While in the foregoing detailed description this invention has beendescribed in relation to certain preferred embodiments thereof, and manydetails have been set forth for purposes of illustration, it will beapparent to those skilled in the art that the invention is susceptibleto additional embodiments and that certain of the details describedherein can be varied considerably without departing from the basicprinciples of the invention.

1. A method of increasing glucose metabolism in an animal, comprisingadministering to an animal (i) a polypeptide comprising an amino acidsequence having at least 95% sequence identity over its entire length tothe amino acid sequence of SEQ ID NO:1 and having the same activity asthe polypeptide of SEQ ID NO:1, or (ii) a polypeptide comprising anamino acid sequence having at least 95% sequence identity over itsentire length to the amino acid sequence of SEQ ID NO:3 and having thesame activity as the polypeptide of SEQ ID NO:3, wherein uponadministration of said polypeptide, glucose metabolism in the animal isincreased.
 2. The method of claim 1, wherein the polypeptide comprisesan amino acid sequence having at least 95% sequence identity over itsentire length to the amino acid sequence of SEQ ID NO:1.
 3. The methodof claim 1, wherein the polypeptide comprises an amino acid sequencehaving at least 95% sequence identity over its entire length to theamino acid sequence of SEQ ID NO:3.
 4. A method of increasinginsulin-mediated glucose uptake in an animal, comprising administeringto an animal (i) a polypeptide comprising an amino acid sequence havingat least 95% sequence identity over its entire length to the amino acidsequence of SEQ ID NO:1 and having the same activity as the polypeptideof SEQ ID NO:1, or (ii) a polypeptide comprising an amino acid sequencehaving at least 95% sequence identity over its entire length to theamino acid sequence of SEQ ID NO:3 and having the same activity as thepolypeptide of SEQ ID NO:3, wherein upon administration of saidpolypeptide, insulin-mediated glucose uptake in the animal is increased.5. The method of claim 4, wherein the polypeptide comprises an aminoacid sequence having at least 95% sequence identity over its entirelength to the amino acid sequence of SEQ ID NO:1.
 6. The method of claim4, wherein the polypeptide comprises an amino acid sequence having atleast 95% sequence identity over its entire length to the amino acidsequence of SEQ ID NO:3.
 7. A method of increasing insulin-stimulatedglucose transport in an animal, comprising administering to an animal(i) a polypeptide comprising an amino acid sequence having at least 95%sequence identity over its entire length to the amino acid sequence ofSEQ ID NO:1 and having the same activity as the polypeptide of SEQ IDNO:1, or (ii) a polypeptide comprising an amino acid sequence having atleast 95% sequence identity over its entire length to the amino acidsequence of SEQ ID NO:3 and having the same activity as the polypeptideof SEQ ID NO:3, wherein upon administration of said polypeptide,insulin-stimulated glucose transport in the animal is increased.
 8. Themethod of claim 7, wherein the polypeptide comprises an amino acidsequence having at least 95% sequence identity over its entire length tothe amino acid sequence of SEQ ID NO:1.
 9. The method of claim 7,wherein the polypeptide comprises an amino acid sequence having at least95% sequence identity over its entire length to the amino acid sequenceof SEQ ID NO:3.
 10. The method of claim 7, wherein said administrationfurther comprises activation of Akt kinase.
 11. The method of claim 10,wherein the activation of Akt kinase comprises an increase inphosphorylation of Akt kinase.
 12. A method of treating a disease orcondition in an animal involving a tissue that contains, uses, orexpresses an omentin polypeptide, comprising administering to an animalin need of treatment a polypeptide comprising an amino acid sequencehaving at least 95% sequence identity over its entire length to theamino acid sequence of SEQ ID NO:1 and having the same activity as thepolypeptide of SEQ ID NO:1, wherein upon administration of thepolypeptide the amount of omentin polypeptide in the tissue of theanimal is increased, and wherein said disease or condition is centralobesity, type 2 diabetes or hyperinsulinemia.
 13. The method of claim12, wherein the tissue comprises stromal vascular cells, pancreas,muscle, liver or adipose tissue.
 14. The method of claim 12, wherein thetissue is adipose tissue.
 15. The method of claim 14, wherein theadipose tissue is omental adipose tissue.
 16. A method of treating adisease or condition in an animal involving a tissue that contains,uses, or expresses an omentin polypeptide, comprising administering toan animal in need of treatment a polypeptide comprising an amino acidsequence having at least 95% sequence identity over its entire length tothe amino acid sequence of SEQ ID NO:3 and having the same activity asthe polypeptide of SEQ ID NO:3, wherein upon administration of thepolypeptide the amount of omentin polypeptide in the tissue of theanimal is increased, and wherein said disease or condition is centralobesity, type 2 diabetes, cardiovascular disease, dyslipidemia,hypertension or hyperinsulinemia.
 17. The method of claim 16, whereinthe tissue comprises stromal vascular cells, pancreas, muscle, liver oradipose tissue.
 18. The method of claim 16, wherein the tissue isadipose tissue.
 19. The method of claim 18, wherein the adipose tissueis omental adipose tissue.